PT - JOURNAL ARTICLE AU - RYUICHI KATO AU - KAZUHIDE IWASAKI AU - HIDEYO NOGUCHI TI - Reduction of Tertiary Amine <em>N</em>-Oxides by Cytochrome P-450 DP - 1978 Jul 01 TA - Molecular Pharmacology PG - 654--664 VI - 14 IP - 4 4099 - http://molpharm.aspetjournals.org/content/14/4/654.short 4100 - http://molpharm.aspetjournals.org/content/14/4/654.full SO - Mol Pharmacol1978 Jul 01; 14 AB - The stimulatory effect of flavins (riboflavin, FMN, and FAD) and viologens (methyl viologen and benzyl viologen) on the anaerobic reduction of tertiary amine N-oxides in liver microsomes was investigated. The reduced flavins or viologens hardly reduced tiaramide N-oxide and imipramine N-oxide in the absence of microsomes. The stimulatory effect of flavins or viologens was completely abolished under an atmosphere of carbon monoxide. FMN or methyl viologen added to the microsomal suspension was rapidly reduced after the addition of NADPH, probably through NADPH-cytochrome c reductase. The reduced FMN or methyl viologen was rapidly reoxidized upon addition of the N-oxides, probably through the oxidation-reduction cycle of cytochrome P-450 initiated by the N-oxides. Although a xanthine oxidase system (xanthine oxidase plus hypoxanthine) did not appreciably reduce cytochrome P-450, tiaramide N-oxide, or imipramine N-oxide, reduction of the N-oxides was markedly stimulated in the presence of flavins or viologens. FMN or methyl viologen in xanthine oxidase-containing microsomal suspensions was rapidly reduced after the addition of hypoxanthine. The reduced FMN or methyl viologen was rapidly reoxidized upon addition of the tertiary amine N-oxides concomitantly with the formation of the corresponding amines. The stimulatory effect of FMN or methyl viologen on the xanthine oxidase-supported N-oxide reduction was dependent on the amount of added xanthine oxidase and was completely blocked under an atmosphere of carbon monoxide. The maximum stimulation by FMN and methyl viologen of NADPH-supported tiaramide N-oxide reduction was about 10- and 100-fold, respectively. The maximal activity of xanthine oxidase-supported N-oxide reductase in the presence of FMN or methyl viologen was about 200 or 6000 nmoles/mg of protein per minute, respectively. Using chemically reduced FMN and methyl viologen as electron donors and tiaramide N-oxide as substrate, maximal reduction rates of 500 and 10,000 nmoles/mg of protein per minute were recorded, respectively. Therefore the maximum turnover number of cytochrome P-450 should be more than 12,000/min. These results indicate that the rate-limiting step in the reduction of N-oxides is the reduction of cytochrome P-450, and other steps seem to be very fast. It was therefore concluded that the mechanism of flavin- or viologen-induced stimulation of NADPH-dependent N-oxide reduction consists of the reduction of flavins or viologens by NADPH-cytochrome c reductase and the reduction of cytochrome P-450 by the reduced form of flavins or viologens. Experiments with purified cytochrome P-450 and NADPH-cytochrome c reductase confirmed this conclusion. ACKNOWLEDGMENT The authors greatly appreciate the kind guidance of Dr. Y. Imai, Osaka University, in the preparation of purified cytochrome P-450 and partially purified NADPH-cytochrome c reductase.